1. Field of the Invention
The present invention relates to aptamers screening method based on graphene without target immobilization in which a single-stranded nucleic acid pool reacts with a non immobilized target material and a single-stranded nucleic acid which has not been bound to the target material or a counter-target material is separated using the graphene, and also relates to specific aptamers selected by the method.
2. Discussion of Related Art
In the conventional biosensor field, an antibody is superior in terms of sensitivity to other various sensing materials used to detect a target material. However, antibodies have a problem in that a process of injecting a target material (antigen) to be detected into an animal's body, obtaining an antibody produced by the animal's biological immune system, and performing a purification process is time-consuming and costly. Further, as compared with aptamers that are rarely limited in target materials, there is a limit in target materials that can be used as antigens. Therefore, it is difficult to manufacture antibodies to low molecular chemical substances such as toxic substances. Typically, an antibody is a very big protein having a size of 100 KDa or more, and thus, when it is used for an electrochemistry-based biosensor, it may be limited in signal detection, and it is remarkably inferior in terms of thermostability to DNA or other chemical substances. Therefore, a conventional technique using an antibody for diagnosing a blood biomarker is not efficient in terms of cost and time, cannot be applied to various fields and is limited in application as a biosensor.
As a novel sensing material to solve such problems, an aptamer which is a nucleic acid construct having a specifically high affinity to various target materials has been used in many studies.
An aptamer is a single-stranded DNA or RNA construct having high specificity and affinity to a specific target. Since an aptamer has a high affinity to a target, has an excellent thermostability and can be synthesized in vitro, it is superior in terms of cost to other sensing materials used in the conventional sensor field. Further, there is no limit in a target material, and thus it is possible to synthesize aptamers with respect to various targets including biomolecules such as proteins, amino acids, etc., small organic chemical substances such as endocrine disrupters or antibiotics, and bacteria. In recent years, due to characteristics of aptamers specifically bound to target materials, many studies on developing an aptamer for application to new drug development, a drug delivery system, and biosensors have been conducted.
The most important factor in a method for developing an aptamer is to distinguish DNA (or RNA) which is bound to a target from DNA which is not bound to the target. To do so, studies on distinguishing DNA by typically immobilizing a target or immobilizing a DNA random library have been conducted. However, the biggest problem of such an immobilization method is that immobilized yield can be low and it is costly and time-consuming to analyze the immobilized yield. Further, the possibility of DNA being directly bound to a separation material (magnetic beads, columns, etc.) used for immobilization cannot be entirely excluded, and the possibility of loss of a DNA pool which may occur when the DNA bound to a target immobilized to the separation material is separated again remains as a limit and problem of the immobilization method. In particular, a low DNA immobilization rate problem which may occur when a DNA library is immobilized is directly related to loss of a DNA pool which is the biggest loss to be avoided during an aptamer development process and thus serves as an upper limit. Moreover, it is difficult to develop aptamers by the immobilization method for heavy metal ions which cannot be immobilized, and thus the immobilization method may be limited in target selection. However, the above-described limits can be overcome by immobilization-free developing technology of aptamer. Further, since a binding site of a target is not limited, it is possible to reduce the number of repetitions of a selection process required for development of an aptamer. Therefore, in order to invent a technique by which an aptamer can be developed through a immobilization-free method, a microelectromechanical system (MEMS), capillary electrophoresis, etc. have been conventionally used, but expensive equipment, complexity in use of devices, necessity of skilled manpower, and the like still remain as problems. Meanwhile, graphene is a two-dimensional carbon structure having excellent thermostability, electrical characteristics, and strength and is bound to a base of a single-stranded DNA by π-stacking, and thus a wide range of studies using such characteristics have been conducted.
Adipokines are proteins secreted from adipocytes and tissues and plays an important role in metabolism. One of adipokine, Nampt (Nicotinamide phosphoribosyltransferase) is a visceral fat-derived protein that has been newly found in recent years and is reported as being closely related to type-2 diabetes caused by obesity. Further, Nampt induces angiogenesis of cancer cells and thus it is an important biomarker associated with colorectal cancer, prostate cancer, stomach cancer, breast cancer, etc. and is also associated with various diseases such as polycystic ovarian syndrome, chronic renal failure, chronic obstructive lung disease, etc.
Meanwhile, bovine viral diarrhea is a disease that causes ulcers in gastrointestinal mucous membranes, diarrhea, respiratory diseases, and death in severe cases. A Bovine Viral Diarrhea Virus type 1 causing bovine viral diarrhea is one of the main threats to the livestock industry. If a pregnant cow is infected, fetal infection occurs highly frequently and various disabilities such as still birth and congenital anomalies occur. A fetus born after being infected at an early gestational stage may carry the virus throughout its life and serve as a source of new infections. In a route of infection with bovine viral diarrhea, infection from a persistently infected cow is considered significant. Therefore, in order to effectively conduct disease surveillance and quarantine activities against bovine viral diarrhea, it is necessary to separate or detect the virus from bovine serum and tissues by a rapid and accurate test method. In this regard, there is reported a method of detecting whether a tissue of a target animal tests positive or negative for the Bovine Viral Diarrhea Virus using an antibody specific to the Bovine Viral Diarrhea Virus epitope (US 2003/0143573). However, conventionally, a method using an antibody for diagnosis has been reported as time-consuming and costly since it is necessary to inject a target material (antigen) to be detected into an animal's body to obtain an antibody produced by the animal's biological immune system, and it is also necessary to perform a purification process. Further, there is a limit in target materials that can be used as antigens. Therefore, it is difficult to manufacture antibodies to toxic substances or viruses. Furthermore, an antibody is a very big protein having a size of 100 KDa or more, and thus, when it is used as an electrochemistry-based biosensor, it may be limited in signal detection, and it is remarkably inferior in terms of thermostability to DNA or other chemical substances, and thus may be limited in application to diagnosis. However, conventionally, there has not been reported an aptamer targeting the Bovine Viral Diarrhea Virus.
The present inventors completed the present invention based on the findings that when graphene is used in developing an aptamer, it is possible to develop an aptamer without a process of immobilizing a target, thereby solving all the problems of the conventional techniques such as limits in binding sites of a target, which may occur when the target is immobilized, and also possible to develop a DNA aptamer without skilled manpower or expensive equipment, and a nucleic acid aptamer capable of firstly diagnosing relevant diseases more accurately and rapidly by accurately measuring a concentration of Nampt, which is a target model, as an important biomarker for type 2 diabetes and cancers, and secondly being specifically bound to a Bovine Viral Diarrhea Virus type 1 only without being bound to other similar substances (BVDV type 2, MDBK cell, Bovine Serum albumin, etc.) for diagnosing bovine viral diarrhea more accurately and rapidly has been developed.